Protective effects of estrogens and caloric restriction during aging on various rat testis parameters

Aim： To investigate the effects of 17β-estradiol （E2）, Peganum harmala extract （PHE） and caloric restriction （CR） on various testis parameters during aging. Methods： Twelve-month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR （40%）. Results： Our results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes： gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats. Conclusion： Besides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species （ROS） produced in large quanti- ties in the aged testis.

Heterologous expression of rat testis GABA_A receptor P3t variant in Chinese hamster ovary cells

Aim: To study the characteristics and possible retention function of specific sequence in the 5'-end of rat testis GABAA receptor β 3t variant. Methods: Rat testis GABAA receptor β 3t variant cDNA was cloned and inserted into two eukaryotic expression vectors of pEGFP-Nl and pEGFP-Cl respectively, which have EGFP reporter gene. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells with the calcium phosphate co-precipitation method. Fluorescence microscope and laser confocal microscope were used to analyze the transfected cells. ConA-Texas-Red was used to label cell endoplasmic reticulum (ER) and study the located area of rat testis β 3t variant in the CHO cells. Results: When the rat testis β 3t variant express in CHO cells, there were two expression pattern, the even and the concentrated expression pattern. Although rat brain β 3 can reach cell membrane, the rat testis β 3t variant can't target to the cell membrane. It may be retained in the ER of CHO cells. Conclusion: There may exist an ER retention signal in the 25 specific amino acid sequence in the N terminal of rat testis β 3t variant.

Preliminary Study on γ-Aminobutyric Acid (GABA_A) Like Receptor in Rat Testis

For making it clear whether GABAA-like receptor is in cells or rat testis and how itsgene expresses in tissue cells, the mRNA of rat testis was microinjected into Xenopusoocyte. The membrane electrobiological results showed that an inward 30 nA micro-currentwas elicited, under two-electrode voltageclamped configuration, by extracellular GABA(500 μmol/L), and both Bicuculline and Picrotoxin could inhibit this effect. It is an indication that the mRNA of rat testis can express GABA receptor in the membrane of Xenonpus oocyte. Using Muscimol as a excitant of GABA receptor type A, the micro--currentwas also elicited to generate; however, using Baclofen as a excitant of GABA receptortype B, the micro-current could not be induced. These results indicate that GABA receptorexpressed in Xenopus oocyte membrane is not type B but for type A. On the basis of the research result above, 24-mer oligonucleotides to be complementary to the high conservativeregion of the cDNAs of neur-GABA receptors were synthesized as probes to hybridize respectively with the RNAs Of brain and testis of adult rat, and of rat testes of the variousages. The dot hybridizations showed that the hybrid signal of rat testis was stronger thanthat of rat brain, meaning that, compared with rat brain, the RNA of rat testis has morehomologous regions with probes, and also implying that the GABA receptor from theRNA Of rat testis may be similar to neural-GABA receptor. This receptor therefore iscalled as GABA_A-like receptor. The dot blot analysis above also showed the testis GABA_Alike receptor. The dot blot analysis above also showed testis GABA_A like receptor mRNAcontent changed with the develOPmental age of rats. There is a very low level of gene expression in the rat testis on day 5 postnatal; there is the highest expression of GABA_A-likereceptor gene in rat testes on day 30to 100 after birth; the expression begins to drop on day200 after birth.

Correlation of expression of preprothyrotropin-releasing hormone and receptor with rat testis development

Objective To investigate the expression regulation of thyrotrophin-releasing hormone (TRH) and TRH receptor (TRH-R), and their role in the development of rat testis.Methods Oligonucleotide primers were designed from the sequences of rat hypothalamus prepro TRH (ppTRH) and pituitary TRH-R cDNA for reverse transcription polymerase chain reaction (RT-PCR). Specific fragments of ppTRH and TRH-R cDNA were cloned and sequenced. Expression plasmids containing ppTRH and TRH-R genes were then constructed, and expression was found in E.coli DH5-α. ppTRH and TRH-R mRNA in the testis was quantitated in RNA samples prepared from rats at different developmental stages by real time quantitative RT-PCR.Results The quantitative analyses demonstrated that no ppTRH and TRH mRNA could be detected at the earliest stage (day 8). ppTRH and TRH mRNA signals were detected on day 15 and increased progressively on days 20, 35, 60 and 90. Conclusion Our results suggest that rat testis could specifically express TRH and TRH-R, and the transcriptions of ppTRH and TRH-R genes in the rat testis were development-dependent. The acquirement of expressed products for ppTRH and TRH-R can be used for further research on the physiological significance of TRH and TRH-R expression in rat testis.

Role of LM23 in cell proliferation and apoptosis and its expression during the testis development

LM23, a gene expressed specifically in the testis in a stage-specific manner, has a diverse range of functions that are important in both the life and death of spermatogenic cells. The aim of this study was to further investigate the expression of LM23 in the developing rat testis and the biological function of LM23 in proliferation and antiapoptosis in vitro. Semiquantitative reverse transcription （RT）-PCR and real-time PCR were used to examine the expression of LM23 in testis at different developmental stages. The results suggested that LM23 mRNA levels in the testis increased progressively after birth. The role of LM23 in proliferation was analyzed with cell counting kit-8 （CCK8）, colony-forming efficiency （CFE） and flow cytometry assays. The results indicated that ectopic expression of LM23 in 293T cells significantly promoted cell proliferation by increasing cell numbers in S phase. Several methods were used, including CCK8, annexin V and propidium iodide staining and western blotting, to determine the role of LM23 in apoptosis. The results showed that LM23 played a protective role in H202-induced apoptosis of 293T cells, mediated at least in part through the Akt/PI3K signal pathway. Taken together, these results provide new insights into the role of LM23 in the development of the testes andspermatogenesis.

HISTOCHEMICAL EFFECT OF THE MULTIGLYCOSIDES OF TRIPTERYGIUM WILFORDII HOOK.F.(GTW)ON THE TESTIS AND EPIDIDYMIS OF RATS

SD Twenty adult male rats were equally divided into a contro1 and an experimcntal group.GTW,10 mg/kg per day,was given to the experimental rats through gastric gavage 6 days a week for 8 weeks.To the control rats,the same amount of vehicle was given The animals were sacrificed when they became infertile and the fol1owing parameters of the testis and epididymis were examined:l)DNA(Feulgen′s method),2)RNA(Brachet′s tech-uique),3)LDH-X(Lojda′s method),4)ATPase(Wachstein′s procedure),5)Succinate dehydrc-genase(SDH,Pearson's method),6)Non-specific esterase(NSE,Lojda's technique)and 7)PAS reaction.Routine examination of the epididymal spermatozoa was also carried out.

Identification of γ-aminobutyric acid transporter (GAT1)on the rat sperm

Some recent studies indicated that GABAergic system is involved in mammalian sperm acrosome reaction (AR), but direct evidence pertaining to the expression of gat1 in mammalian sperm is not yet demonstrated. In this study, we evaluated the presence of 67kDa GAT1 protein and mRNA in rat testis by Western blotting and reverse transcription-polymerase chain reaction. Meanwhile, immunohistochemical and immunofluorescent analyses also identified GAT1 protein on the elongated spermatid and sperm. These results indicated that rat testis is a novel site of gat1 expression. Further studies should be taken to explore the role of GAT1 protein on sperm acrosome reaction.

Age-related decrease in aromatase and estrogen receptor （ERα and ERβ） expression in rat testes： protective effect of low caloric diets

Aim： To examine the effects on rat aging of caloric restriction （CR1） and undernutrition （CR2） on the body and on testicular weights, on two enzymatic antioxidants （superoxide dismutase and catalase）, on lipid peroxidation and on the expression of testicular aromatase and estrogen receptors （ER）. Methods： CR was initiated in 1-month-old rats and carried on until the age of 18 months. Results： In control and CR2 rats an age-related decrease of the aromatase and of ER （α and β） gene expression was observed; in parallel a diminution of testicular weights, and of the total number and motility of epididymal spermatozo was recorded. In addition, aging in control and CR2 rats was accompartied by a significant decrease in testicular superoxide dismutase, catalase activities, and an increase in lipid peroxidation level （thiobarbituric acid reactive substance）, associated with alterations of spermatogenesis. Conversely, caloric restriction-treatment exerted a protective effect and all the parameters were less affected by aging. Conclusion： These results indicate that during aging, a low caloric diet （not undernutrition） is beneficial for spermatogenesis and likely improves the protection of the cells via an increase of the cellular antioxidant defense system in which aromatase/ ER could play a role. （Asian J Andro12008 Mar; 10： 177-187）

Cloning of rat sp56,the homologue of mouse sperm ZP3 receptor-sp56

Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3 (mZP3) receptor. Up to date, its homologue has only been cloned from guinea pig. namely AM67. Based on the cDNA sequence of mouse sp56, we designed a pair of primer to amplify its homologue from rat testis cDNA. Using RT-PCR, two fragments of 743 bp and 938 bp were amplified. The PCR products show very high homology to mouse sp56. However, the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56. Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues, Northern blot shows that a -2.0 kb mRNA expresses specifically in testis. Employed the RACE method, two full cDNA sequences of rat sp56 were obtained. A Mr -42 KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method. Rat sp56 was localized on rat sperm head by the indirect immunofluorescence method. Rat sp56 immunoreactivity was detected from the early pachytene spermatocytes and throughout the spermatogenesis. Its cloning will further our understanding of the mechanism of the sperrn-egg recognition and binding.

Isolation and Purification of Rat Testicular Lactate Dehydrogenase C_4

The lactate dehydrogenase isoenzyme C4 ofrat testicle was purified with a yield of 1 2.3% anda raise of specific activity to 784-fold.The purified product was PAGE homogeneous and migrated afterLDH3 during electrophoresis.

Further Analysis of El Tor Vibrio Cholerae Strains 1d:Comparison of Two Typing Methods

The lactate dehydrogenase isoenzyme C4 ofrat testicle was purified with a yield of 1 2.3% anda raise of specific activity to 784-fold.The purified product was PAGE homogeneous and migrated afterLDH3 during electrophoresis.